The hypothalamic-pituitary axis (HP) is crucial regulator of endocrine function. Disruption of this system during development occurs in diseases such as hypopituitarism (1 in 4,000 live births), and has serious consequences for human health. Symptoms vary depending on which hormones are affected, but can include abnormal sexual maturity, growth insufficiency, inability to thermoregulate and infertility. Many cases of hypopituitarism also present with an ectopic posterior pituitary (EPP), which contains the terminal axons of hypothalamic arginine vasopressin (AVP) neurons. In fact, human mutations of HESX1, LHX4 and SOX3 result in EPP as well as hypopituitarism. EPP patients can also develop central diabetes insipidus (DI), caused by AVP deficiency. Hypothalamic neurons that project to the anterior lobe through the median eminence (ME), such as somatostatin (SS), are affected by both hypopituitarism and multiple anterior pituitary hormone deficiency (MPHD; 1:4000-1:10,000 live births). Although known mutations account for some cases of hypopituitarism, MPHD and EPP, the genetic basis of these devastating diseases is largely unknown. In fact, the molecular pathways that mediate functional hypothalamic development are not well understood. We do know that proper development of hypothalamic neurons requires extrinsic signaling, which initiates distinct spatial and temporal patterns of transcription factor expression, specifying neuroendocrine cell fate. The Notch signaling pathway is an evolutionarily conserved mechanism that regulates cell proliferation and differentiation in the development of many systems. This pathway is present in the developing HP, but its function in neuroendocrine cell differentiation is not known. Using transgenic and knockout mice, the proposed studies will determine if Notch signaling, globally and specifically within the hypothalamus, is necessary and sufficient for hypothalamic cell differentiation and axon targeting to the pituitary. Using a novel mass spectrometry based approach, pituitaries from these mouse models will be analyzed for differences in neuropeptide content. Results from these studies will provide insight into the role of Notch signaling in the functional development of hypothalamic neurons and may reveal genetic causes of congenital HP dysfunction.